Chloride bound to oxidized cytochrome c oxidase controls the reaction with nitric oxide

The reaction of nitric oxide (NO) with oxidized fast cytochrome c oxidase was investigated by stopped-flow, amperometry, and EPR, using the enzyme as prepared or after 'pulsing'. A rapid reduction of cytochrome a is observed with the pulsed, but not with the enzyme as prepared. The reactive species (λ(max) = 424 nm) reacts with NO at k = 2.2 x 10⁵ M^-1 s^-1 at 20 °C and is stable for hours unless Cl^- is added, in which case it decays slowly (t( 1/2 ) ~ 70 min) to an unreactive state (λ(max) = 423 nm) similar to the enzyme as prepared. Thus, Cl^- binding prevents a rapid reaction of NO with the oxidized binuclear center. EPR experiments show no new signals within 15 s after addition of NO to the enzyme as prepared. Amperometric measurements show that the pulsed NO-reactive enzyme reacts with high affinity and a stoichiometry of 1 NO/aa₃, whereas the enzyme as prepared reacts to a very small extent (<20%). In both cases, the reactivity is abolished by pre-incubation with cyanide. These experiments suggest that the effect of 'pulsing' the enzyme, which leads to enhanced NO reactivity, arises from removing Cl^- bound at the oxidized cytochrome a₃-Cu(B) site.